Despite similar nutrient and hormone exposure, there is wide variation in lipid content and function of different fat depots within the same individual. The fat depot from which preadipocytes are cultured affects their capacity to differentiate, even in cells cultured for several cell generations and studied at the single cell level. Thus, mechanisms intrinsic to adipose cells contribute to interdepot variation in fat tissue function, in addition to variation among depots due to extrinsic factors, such as vascular supply and innervation. We found that expression of C/EBPalpha was blunted during differentiation in epdidymal preadipocytes, while that of C/EBPbeta was not. Transient transfection to increase C/EBPalpha expression in epididymal preadipocytes enhanced their ability to differentiate. This implicates events upstream of C/EBPalpha but downstream of C/EBPbeta during differentiation as the cause of interdepot variation in preadipocyte differentiation. PPARgamma is expressed after C/EBPbeta and promotes C/EBPalpha expression. Thiazolidinediones, which act by binding to PPARgamma are less effective in inducing epididymal than perirenal preadipocyte differentiation. TNFalpha blunts C/EBPalpha expression and blocks effects of PPARgamma. We found that epididymal preadipocytes release more TNFalpha than perirenal cells, and that exposing differentiating perirenal cells to concentrations equivalent to those in epididymal cultures blunted their differentiation. Based on our preliminary findings, our hypothesis is that regional variation in TNFalpha release and PPARgamma activity plays a central role in effects of fat depot origin on ability of preadipocytes to express C/EBPalpha and differentiate. We propose the following specific aims to test this hypothesis. Aim 1 is to establish the role of TNFalpha in regional variation of preadipocyte differentiation and C/EBPalpha expression. Aim 2 is to determine the role of PPARgamma and related factors in causing variation among depots in preadipocyte differentiation, C/EBPalpha expression, and TNFalpha release. Aim 3 is to assess the relevance of our findings to humans by determining if preadipocyte C/EBPalpha and PPARgamma expression and TNFalpha production differ between human omental and abdominal subcutaneous preadipocytes from the same subjects. The studies we propose to understand the mechanism(s) underlying interdepot variation may add not only to knowledge about effects of tissue site on the capacity of progenitors within that tissue to differentiate, but also about why fat tissue mass, metabolism, hormone responsiveness, and function vary among depots. These studies will point to potential future strategies for altering function in specific fat depots.